Fig 2: The 3'-UTR is involved in downregulation of PXR expression and decreased rifampicin-mediated upregulation of CYP3A4. (A) HepG2 cells were transiently transfected with either pmiRGLO-UTR or empty pmiRGLO vector (50 ng). After 48 h, the samples were analyzed by the Dual-Luciferase Reporter Assay kit. Firefly luciferase activities were normalized to Renilla luciferase activities. The data are shown as the mean ± SD (n = 3) and are expressed as relative change to an empty pmiRGLO vector defined as 100%. HepG2 cells were transiently transfected with either pSG5-PXR or pSG5-PXR-UTR vector (200 ng) and cotransfected with pSG5-RXRa (200 ng) for 24 h. After another 24 or 48 h in fresh medium, total RNA was isolated and analyzed for basal levels of NR1I2 mRNA by RT-qPCR (B) or for PXR protein expression (C). The HPRT reference gene-normalized data are shown as the mean ± SD (n = 3) and are expressed as relative change to values gained from pSG5-PXR/pSG5-RXRa experiments (100%). Cell lysates were analyzed for expression of PXR protein by Western blotting. ß-Actin was used as a loading control. Representative Western blotting results are shown. (D) HepG2 cells were transiently transfected with either pSG5-PXR or pSG5-PXR-UTR vector (200 ng) and cotransfected with pSG5-RXRa (200 ng). The next day, cells were treated with rifampicin (Rif, 10 µmol/L) for 24 h. Total RNA was isolated and analyzed for induction of CYP3A4 mRNA by RT-qPCR. The HPRT reference gene-normalized data are shown as the mean ± SD (n = 3) and are expressed as fold change compared to vehicle-treated controls (defined as 1). (E) COS-1 cells were transiently transfected with a CYP3A4 promoter luciferase reporter vector (p3A4-luc, 150 ng), pRL-TK (30 ng), and pSG5-RXRa (50 ng) together with either pSG5-PXR, pSG5-PXR-UTR or empty pSG5 vector (50 ng). The next day, cells were treated with rifampicin (Rif, 10 µmol/L) or vehicle (DMSO; 0.1%) for another 24 h. Then, samples were analyzed using a Dual-Luciferase Reporter Assay. Firefly luciferase activities were normalized to Renilla activities. The data are shown as the mean ± SD (n = 3) and are expressed as fold change compared to respective DMSO-treated controls. (F) COS-1 cells were transiently transfected with either a pSG5-PXR or pSG5-PXR-UTR construct (200 ng) and cotransfected with pSG5-RXRa (200 ng). The next day, cells were treated with vehicle (DMSO; 0.1%) or rifampicin (Rif, 10 µmol/L) for 48 h. Samples were analyzed for expression of PXR protein by Western blotting. ß-Actin was used as a loading control. Representative Western blotting results are shown. *P < 0.05, **P < 0.01 indicate statistically significant effects of the 3'-UTR region of NR1I2 mRNA. (G). Schematic overview showing sequence complementarity between particular miRNAs and in silico predicted or experimentally confirmed miRNA-response elements (MREs) within the 3'-UTR of human NR1I2 mRNA. Green letters depict seed regions of miRNAs. Blue letters refer to sequences within MREs complementary to miRNA seed regions. Numbers above sequences indicate a position of nucleotides downstream from the coding region of NR1I2 mRNA.

Fig 3: Ligand-dependent activation of PXR downregulates NR1I2 expression in primary human hepatocytes. NR1I2 mRNA (A) or CYP3A4 mRNA (B) expression in primary human hepatocytes (donor Liv13) treated with vehicle (DMSO; 0.1%, v/v) or rifampicin (Rif, 10 µmol/L) for 12, 24, and 48 h, respectively. The expression data were normalized to the RPLP0 reference gene, and data are expressed as relative change to corresponding control (DMSO-treated) experiments defined as 100%. (C) Western blotting analysis of PXR, CYP3A4 and RXRa expression in primary human hepatocytes (Liv13) treated with vehicle (DMSO; 0.1%) or Rif (10 µmol/L) for 24 or 48 h. ß-Actin was used as a loading control. (D) NR1I2 mRNA and (E) CYP3A4 mRNA expression. Primary human hepatocytes (Liv1–13) and commercial (Liv14) human hepatocyte cultures were treated with vehicle (DMSO; 0.1%), Rif (10 or 5 µmol/L), SR-12813 (3 µmol/L) or phenobarbital (500 µmol/L) for 24 h. The data are presented as fold change in expression after PXR ligand treatment versus vehicle (DMSO)-treated controls defined as 1. Nonparametric Wilcoxon matched-pairs tests or paired t test were used to statistically compare paired expression data between control and PXR ligand-treated samples in human hepatocytes. *P < 0.05, statistically significant effect of PXR ligand (paired t test, P-value in parentheses).

Fig 4: Induction of CAR target genes in primary human hepatocyte models and interactions of selected candidates with CAR transcription variants and mouse CAR. (A) PHHs were treated with compounds 37, 39, 40, 41, and 42 together with CITCO for 24 h. The expression of CYP2B6 mRNA, a prototype CAR target gene, was analyzed using RT-qPCR in technical triplicates. Data are presented as mRNA fold induction to control (vehicle-treated) samples. (B) Translocation experiments with EGFP-hCAR + Ala chimera in COS-1 cells treated with tested compounds (10 µM) for 24 h before confocal microscopy. Data are presented as % of cells with specific cytoplasm or mixed/nuclear localization of pEGFP-hCAR + Ala chimeric protein. (C) Interactions of compounds 37, 39, 40, 41, and 42 with human wtCAR in the CAR LBD assembly assay (wtCAR AA) or with wtCAR inhibited with PK11195 (0.1 µM), with CAR2 or CAR3 variants, or with mouse Car (mCar). (D) PHHs from five donors were treated with compound 39 and CITCO (1 and 10 µM, respectively) for 48 h. CAR target genes CYP2B6, CYP3A4, and CYP2C9 mRNA expression have been studied using RT-qPCR. Data are presented as fold induction to control (vehicle-treated) samples. Western blotting experiments with primary human (BioIVT) treated with comp. 39, rifampicin (rif), CITCO, and PXR antagonist SPA70 (10 µM) for 48 h. Monoclonal anti-CYP2B6 antibody (PA5-35032) was used to detect CYP2B6 protein. (E,F) HepaRG cells and HepaRG KO CAR cells without functional CAR activity were treated with phenobarbital (500 µM), CITCO, rifampicin (10 µM), or compound 39 at a 1 µM concentration for 48 h. CYP2B6 and CYP3A4 mRNA expression have been analyzed using RT-qPCR. (G) LS174T cells expressing PXR, but without functional CAR, were treated with compound 39, rifampicin (rif), CITCO, SPA70, or compound 39 at a 10 µM concentration for 48 h. CYP2B6 and CYP3A4 mRNA expression has been analyzed using RT-qPCR. (H) Luciferase gene reporter assay with the CYP3A4 gene promoter construct (p3A4-luc) in HepG2 cells transfected with either PXR or CAR3 expression constructs. Cells were treated for 24 h before analysis. (I) Dose–response activation of CAR2 and CAR3 variants with compound 39 in luciferase reporter gene assays. *p < 0.05 and **p < 0.01-significant CYP2B6 or CYP3A4 mRNA upregulation, p3A4-luc activation or EGFP-hCAR + Ala fusion protein nuclear translocation to control samples; f-statistically significant effect of SPA70 on rifampicin-mediated CYP3A4 mRNA expression or activation of the p3A4-luc luciferase construct.

Fig 5: Plasma and microsomal stability experiments and single-dose pharmacokinetics in C57BL/6N mice. The stability of compound 39 in human plasma (A) and human microsomes with S9 fraction (B) were analyzed after 2 h of treatment. (C) Pharmacokinetics (PK) after single-dose application of compound 39 as hydrochloric salt either via i.v. or peroral application (10 mg/kg, n = 4) were analyzed in mice over 480 min. (D) Metabolites M1 (comp. 41), M2 (comp. 40), and M3 (comp. 34, 2-(4-chlorophenyl)-3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine) of compound 39 were observed after i.v. application. Samples have been analyzed using HPLC-MS/MS. (E) Inhibition of CYP3A4, CYP2B6, and CYP1A2 enzymes in microsomes. Compound 39 was tested in the concentration range from 0.1 nM up to 30 µM. Relative activity data were fitted, and dose–response curves were used to obtain IC50.